Generally in the technology of manipulating genetic material or in evaluating the genetic character of an organism, it is often desirable to ascertain if a particular gene or part of a gene is present in an organism or in an extracellular extract of genetic material from an organism. Since any gene or gene portion is, in essence, a specific sequence of nucleotide bases forming all or part of a polynucleotide molecule, to discover if a particular gene or gene fragment is present in a sample, it is possible to directly test the sample polynucleotide to discover if the specific sequence of nucleotide bases forming the gene is present in the sample.
The present method generally used for detection of a specific nucleotide target sequence in a DNA or RNA polynucleotide sample depends upon the hybridization of a sample, referred to as a test sequence, to a radioactively labeled complementary DNA probe containing the target sequence. The cDNA probe may be isolated in quantity or may be cloned using known techniques, such as by insertion into a DNA vector, either a plasmid or a phage, which is inserted into a bacterium propagated in a nutrient medium. If the media includes a radioactive isotope which is absorbed by the bacterium into its own DNA, such as .sup.32 P, the cloned DNA probe includes a radioactive component therein. Alternatively, the isolated DNA probes may also be labeled radioactively by in vitro chemical reaction. In the detection procedure, the test sequence, which is typically immobilized on a filter, is exposed to the radioactively labeled complementary DNA probe, after which the test sequence is then washed. A radioactive assay of the test sequence is then conducted by a scintillation spectrometer or by autoradiography. The amount of radioactive isotope detected by the radioactive assay is indicative of the amount of the radioactive cDNA probe which has been hybridized to the test sequence, and is therefore an indication of whether the test sequence includes the target sequence which is sought.
While assay methods utilizing radioactively-tagged polynucleotides are effective for the assay of such sequences, there are several disadvantages inherent in such procedures. The radioactive materials which are required are inherently somewhat hazardous, and are also inherently unstable. Furthermore a laboratory performing such an assay procedure must be specially licensed and have specially trained technicians in order to use radioactive materials. Many laboratories may be unable to conduct such assays because of the burdensome nature of the safeguards encumbent in the use of such materials and the special licensing procedure required by it. The assay method of the present invention is, in part, intended to avoid the use of radioactive materials.